Large-scale fish oil EPA separation and ethyl esterification method

Abstract

The invention relates to a large-scale fish oil EPA (eicosapentaenoic acid) separation and ethyl esterification method, and belongs to the technical field of fish oil EPA separation. S2, purifying the EPA; s3, EPA ethyl esterification: adding the purified EPA into a certain amount of absolute ethyl alcohol, slowly dropwise adding a small amount of concentrated sulfuric acid under the condition of continuous stirring, controlling the reaction temperature and time, cooling to room temperature after the reaction is finished, then adjusting the pH value to 7 by using a 2M sodium hydroxide solution, washing an organic phase by using distilled water, drying by using anhydrous sodium sulfate, and performing vacuum concentration to obtain an EPA ethyl ester crude product; and then carrying out molecular distillation to obtain the high-purity EPA-EE. Compared with the prior art, the technical scheme provided by the invention has the advantages that efficient separation of EPA and DHA can be realized under conventional industrial equipment, and a high-purity EPA single product is prepared; besides, EPA is further subjected to ethyl esterification treatment, so that the stability and bioavailability of EPA are remarkably improved, the separation cost is greatly reduced, and the production efficiency is also improved.

Description

Technical Field

[0001] The present invention relates to the technical field of fish oil EPA separation, and particularly to a method for large-scale separation and ethyl esterification of fish oil EPA. Background Art

[0002] Fish oil is rich in ω-3 polyunsaturated fatty acids, mainly including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). EPA and DHA have been widely studied and applied due to their benefits in improving cardiovascular health, anti-inflammatory, promoting brain function, and supporting the immune system. Among them, EPA has been proven to have functions such as reducing triglycerides and cholesterol in the human body and preventing thrombosis, which helps to reduce the prevalence of cardiovascular diseases. With the continuous improvement of people's health awareness and the wide recognition of the efficacy of fish oil, the global fish oil market scale has been continuously growing. According to market research data, the annual growth rate of the fish oil market remains between 5% and 7%. Especially in the health care products and pharmaceutical fields, the demand for high-purity EPA is increasing day by day. In order to fully exert the pharmacological effects of EPA, it is crucial to prepare high-purity EPA. At the same time, due to the characteristics of polyunsaturated fatty acids, high-purity EPA is often unstable. Therefore, most manufacturers often convert fish oil into ethyl ester form. Currently, Vascepa (96% purity EPA-EE, without DHA) produced by Amarin Corporation is the only EPA drug approved by the US FDA for marketing and is used as an adjunct to statin therapy. However, the preparation of high-purity EPA on an industrial scale faces significant technical challenges. The main challenge lies in the fact that the molecular structures of EPA and DHA are similar and the polarity difference is extremely small. Traditional separation methods are difficult to effectively distinguish these two fatty acids and it is difficult to obtain high-purity EPA. Therefore, the development of industrial technologies that can effectively separate EPA and DHA has become a research hotspot.

[0003] At present, the technologies for separating EPA and DHA industrially mainly include chromatographic separation, molecular distillation, membrane separation, etc. Although these methods can achieve the separation of EPA and DHA to a certain extent, there are still many deficiencies. Taking chromatographic separation as an example, although chromatography can achieve the separation of EPA and DHA, its cost is high, the operation is complex, and the yield is low, which is not suitable for large-scale industrial production. Molecular distillation separates based on the volatility differences of different fatty acids. However, due to the small boiling point differences between EPA and DHA, the purity is often difficult to reach the ideal state. Membrane separation technology separates based on the molecular size and solubility differences of different fatty acids. Although the operation is relatively simple, the membrane materials are prone to aging and pollution, and the separation efficiency and purity still need to be improved. There have been many Chinese invention patents proposing different methods for separating EPA and DHA. For example, Chinese Patent CN103962091A proposes a method for separating EPA and DHA using silver ion-modified amino silica gel. This method improves the separation efficiency by modifying and filling the stationary phase based on a chromatographic column. However, the preparation of the filler for this method is expensive, the separation efficiency is low, and it is not suitable for large-scale industrial production. Chinese Patent CN108164415B introduces a method for separating EPA and DHA by high-speed countercurrent chromatography, but this method requires high equipment investment and complex operation processes, making it difficult to achieve industrialization. Although these existing separation technologies can achieve good separation effects in a laboratory environment, there are problems such as expensive equipment, complex operation, low output, and insufficient purity in industrial applications. Summary of the Invention

[0004] To overcome the above-mentioned defects of the prior art, the present invention provides a method for large-scale separation and ethyl esterification of EPA from fish oil to solve the problems raised in the background art.

[0005] To achieve the above-mentioned invention object, the present invention provides a method for large-scale separation and ethyl esterification of EPA from fish oil. S1: Prepare iodolactonide

[0006] Dissolve the polyunsaturated fatty acid mixture in tetrahydrofuran and continuously stir. Subsequently, add sodium bicarbonate and an aqueous solution of sodium iodide to the above mixture and continue stirring. Then continue to add elemental iodine, and react to form iodolactone 3 and iodolactone 4;

[0007] Subsequently, a certain amount of sodium bicarbonate and a certain amount of sodium sulfite were weighed and dissolved in distilled water to form a reaction solution, and the above mixture was slowly poured into the reaction solution to quench the reaction. The obtained mixture was extracted with ethyl acetate, and the organic phase was distilled under reduced pressure to obtain a mixture containing iodolactone 3 and iodolactone 4. The mixture was further dissolved in n-hexane, washed with a 0.5 M sodium carbonate alkaline solution, and the separated n-hexane was washed with saturated brine, dried over anhydrous sodium sulfate, and then distilled under reduced pressure. The obtained solution was passed through a sintered funnel with a silica gel powder layer spread on the surface, and the filtrate was distilled under reduced pressure to obtain purified iodolactone 3 and iodolactone 4;

[0008] S2: Purification of EPA

[0009] Under nitrogen protection, the above purified iodolactone 3 and iodolactone 4 were added to an acetonitrile solution containing sodium iodide or a chloroform solution containing sodium iodide, and then triethylsilane was added. The mixture was stirred at room temperature. After the reaction was completed, a certain amount of sodium sulfite and a certain amount of sodium citrate were weighed and dissolved in an aqueous solution, and this solution was added to the solution after the above reaction to terminate the reaction. Subsequently, it was extracted with n-hexane-dichloromethane, the extract was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The obtained concentrated solution was dissolved in n-hexane, then extracted with a 0.5 M sodium carbonate solution, washed with a 1:1 n-hexane-ether solution. After the washing was completed, it was acidified with 4.5 M hydrochloric acid, then extracted with a 2:1 n-hexane-ether, and then washed with saturated brine and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain pure EPA. The purified pure EPA was structurally identified by NMR;

[0010] S3: Esterification of EPA

[0011] The above pure EPA was added to a certain amount of absolute ethanol, and a small amount of concentrated sulfuric acid was slowly added dropwise under continuous stirring, controlling the reaction temperature and time. After the reaction was completed, it was cooled to room temperature, and then the pH value was adjusted to 7 with a 2 M sodium hydroxide solution. The organic phase was washed with distilled water, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product of EPA ethyl ester. Subsequently, molecular distillation was carried out to obtain high-purity EPA-EE, and its structure was identified by NMR.

[0012] Furthermore, the preparation method of the sodium iodide aqueous solution includes: dissolving 110 - 170 g of sodium iodide in 850 - 1300 mL of distilled water and stirring and mixing.

[0013] Furthermore, both the sodium carbonate alkaline solution in step S1 and the sodium carbonate solution in step S2 include 1:1 methanol and water.

[0014] Furthermore, in step S3, the reaction temperature is controlled at 60°C - 70°C, and after reacting for 3 - 5 hours, it is cooled to room temperature.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] Compared with the prior art, the technical solution of the present invention can achieve efficient separation of EPA and DHA under conventional industrial equipment, and prepare high-purity EPA single product; in addition, by further ethyl esterification of EPA, not only the stability and bioavailability are significantly improved, but also the separation cost is greatly reduced, the production efficiency is improved, and its application in drugs and health products is more extensive. The technical solution of the present invention not only significantly reduces the separation cost, but also improves the production efficiency, and has broad market prospects and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the high-purity EPA separation and its ethyl esterification route of the present invention;

[0018] Figure 2 For the pure EPA of the embodiment of the present invention 1 1H NMR spectrum;

[0019] Figure 3 For the pure EPA of the embodiment of the present invention 13 13C NMR spectrum;

[0020] Figure 4 For the EPA-EE of the embodiment of the present invention 1 1H NMR spectrum;

[0021] Figure 5 For the EPA-EE of the embodiment of the present invention 13 13C NMR spectrum. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the invention, rather than all of the embodiments. The embodiments of the present invention will be described below with reference to the accompanying drawings.

[0023] Example 1

[0024] A large-scale method for separating and ethyl esterifying EPA from fish oil, comprising the following steps:

[0025] 1. Preparation of iodolactonide:

[0026] Dissolve 100 g of the polyunsaturated fatty acid mixture in a flask containing 2 L of tetrahydrofuran and continuously stir. Subsequently, add 70 g of sodium bicarbonate and an aqueous sodium iodide solution to the mixture. The aqueous sodium iodide solution is prepared by dissolving 114 g of sodium iodide in 875 mL of distilled water. After stirring for 20 minutes, continue to add 390 g of elemental iodine to the flask. The system is then reacted at 4 °C for 48 hours. At this time, the polyunsaturated fatty acids EPA1 and DHA2 undergo an iodine-mediated lactonization reaction to form iodolactone 3 and iodolactone 4. Subsequently, weigh 640 g of sodium bicarbonate and 450 g of sodium sulfite and dissolve them in 2 L of distilled water, and slowly pour the mixture into the reaction solution to quench the reaction; the resulting mixture is extracted with ethyl acetate, and the organic phase is distilled under reduced pressure to obtain a mixture containing iodolactone 3 and iodolactone 4. The crude mixture is dissolved in 3 L of n-hexane, washed with 1.5 L of 0.5 M sodium carbonate methanol-water alkaline solution, with a methanol-water ratio of 1:1. The separated n-hexane is washed with saturated brine, dried over anhydrous sodium sulfate, and then distilled under reduced pressure. The resulting solution passes through a sintered funnel with a 300-mesh silica gel powder layer spread flat on the surface with a height of 5 cm. After the filtrate is distilled under reduced pressure, 42 g of purified iodolactone 3 and iodolactone 4 are obtained;

[0027] 2. Purify EPA:

[0028] Under nitrogen protection, add the above 42 g of purified iodolactone 3 and iodolactone 4 to a 500 mL acetonitrile solution containing 64 g of sodium iodide. Subsequently, add 18 mL of 2-methyl-2-butene solution and 14.4 mL of triethylsilane, and the mixture is stirred at room temperature for 2 hours. Due to the structural difference, the γ-lactone ring in iodolactone 4 is more stable than the δ-lactone ring in iodolactone 3. Therefore, this process can selectively cleave iodolactone 3 to regenerate EPA, while iodolactone 4 does not react. After the reaction is completed, weigh 14.4 g of sodium sulfite and 13.5 g of sodium citrate and dissolve them in 1300 mL of aqueous solution, and add this solution to the system to terminate the reaction. Subsequently, the mixture is extracted with 4:1 n-hexane-dichloromethane, and the extract is washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated. The resulting concentrate is dissolved in 1200 mL of n-hexane, and then extracted with 650 mL of 0.5 M sodium carbonate methanol-water alkaline mixed solution, with a methanol-water ratio of 1:1. Discard the n-hexane layer, and the alkaline aqueous solution is washed with 1:1 n-hexane-ether. At this time, iodolactone 4 is dissolved in the n-hexane-ether mixture and removed, and EPA is dissolved in the alkaline aqueous solution in the form of a soap. After washing, acidify with 150 mL of 4.5 M hydrochloric acid aqueous solution, extract with 2:1 n-hexane-ether, wash the organic phase with saturated brine and dry over anhydrous sodium sulfate, and then concentrate under reduced pressure to obtain 16.5 g of pure EPA. This compound is then structurally characterized using nuclear magnetic resonance ( 1 H NMR, 13 C NMR), and the spectral results are as Figure 2 、Figure 3 as shown;

[0029] EPA(1): 1 H NMR (300 MHz, Chloroform-d) δ 5.50–5.22 (m, 10H), 2.83 (dt, J = 10.9, 5.5 Hz, 8H), 2.37 (t, J = 7.5 Hz, 2H), 2.20–1.99 (m, 4H), 1.79–1.63 (m, 2H), 0.97 (t, J = 7.5 Hz, 3H). 13C NMR (75 MHz, Chloroform-d) δ 180.16, 132.05, 129.04, 128.77, 128.58, 128.27, 128.18, 128.09, 127.89, 127.03, 33.44, 26.46, 25.64, 25.55, 24.49, 20.58, 14.30.

[0030] 3. Esterification of EPA:

[0031] Add 16.5 g of the above pure EPA to 50 mL of absolute ethanol. While stirring continuously, slowly add 1 mL of concentrated sulfuric acid to the solution. Then, control the reaction temperature between 60 °C and 70 °C. After 4 hours, cool to room temperature. Subsequently, neutralize the system to pH = 7 using 2M sodium hydroxide solution. Then, wash the organic phase with distilled water, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the crude product of EPA ethyl ester. Subsequently, perform molecular distillation to obtain 15.8 g of high-purity EPA-EE. This compound was then structurally characterized using nuclear magnetic ( 1 H NMR, 13 13C NMR), and the spectral results are as Figure 4 , Figure 5 shown.

[0032] EPA-EE: 11H NMR (300 MHz, Chloroform-d) δ 5.47–5.25 (m, 10H), 4.12 (q, J = 7.1 Hz, 2H), 2.91–2.73 (m, 8H), 2.30 (t, J = 7.5 Hz, 2H), 2.18–2.00 (m, 4H), 1.79–1.59 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H), 0.97 (t, J = 7.5 Hz, 3H). 13C NMR (75 MHz, Chloroform-d) δ 173.62, 132.03, 129.02, 128.79, 128.56, 128.25, 128.10, 127.87, 127.02, 60.23, 33.72, 26.57, 25.63, 25.61, 25.54, 24.81, 20.57, 14.29, 14.26。

[0033] Example 2

[0034] A large-scale method for separating and ethylating EPA from fish oil, comprising the following steps:

[0035] 1. Preparation of iodo lactonide:

[0036] Dissolve 150 g of the polyunsaturated fatty acid mixture in a flask containing 3 L of tetrahydrofuran and stir continuously. Subsequently, add 130 g of sodium bicarbonate and an aqueous sodium iodide solution to the mixture, where the aqueous sodium iodide solution is prepared by dissolving 70 g of sodium iodide in 1300 mL of distilled water. After stirring for 20 minutes, continue to add 420 g of elemental iodine to the flask. The system is then reacted at 4°C for 48 hours. At this time, the main polyunsaturated fatty acids EPA 1 and DHA 2 undergo an iodine-mediated lactonization reaction to form iodo lactone 3 and iodo lactone 4. Subsequently, weigh 960 g of sodium bicarbonate and 675 g of sodium sulfite and dissolve them in 3 L of distilled water, and slowly pour the mixture into the reaction solution to quench the reaction. The resulting mixture is extracted with ethyl acetate, and the organic phase is distilled under reduced pressure to obtain a mixture containing iodo lactone 3 and iodo lactone 4. Dissolve the mixture in 4 L of n-hexane, wash it with 2 L of 0.5 M sodium carbonate methanol-water basic mixed solution, and the methanol-water ratio is 1:1. The separated n-hexane is washed with saturated brine, dried over anhydrous sodium sulfate, and then distilled under reduced pressure. The resulting solution passes through a sintered glass funnel with a 5 cm high layer of 300-mesh silica gel powder spread on the surface. After the filtrate is distilled under reduced pressure, 60.9 g of refined iodo lactone 3 and iodo lactone 4 are obtained;

[0037] 2. Purification of EPA:

[0038] Under nitrogen protection, 60.9 g of the above-mentioned refined iodolactones 3 and 4 were added to 750 mL of a chloroform solution containing 100 g of sodium iodide. Subsequently, 27 mL of 2-methyl-2-butene solution and 21.6 mL of triethylsilane were added, and the mixture was stirred at room temperature for 2 hours. Due to the structural difference, the γ-lactone ring in iodolactone 4 is more stable than the δ-lactone ring in iodolactone 3. Therefore, this process can selectively cleave iodolactone 3 to regenerate EPA, while iodolactone 4 does not react. After the reaction, 21.6 g of sodium sulfite and 20.3 g of sodium citrate were weighed and dissolved in 2000 mL of aqueous solution, and this solution was added to the system to terminate the reaction. Subsequently, the mixture was extracted with n-hexane-dichloromethane at a ratio of 4:1, and the extract was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The resulting concentrated solution was dissolved in 1500 mL of n-hexane, and then extracted with 1000 mL of a 0.5 M sodium carbonate methanol-water alkaline mixed solution with a methanol-water ratio of 1:1. The n-hexane layer was discarded, and the alkaline aqueous solution was washed with n-hexane-ether at a ratio of 1:1. At this time, iodolactone 4 was dissolved in the n-hexane-ether mixture and removed, and EPA was dissolved in the alkaline aqueous solution in the form of soap. After washing, the solution was acidified with 200 mL of 4.5 M hydrochloric acid to obtain an aqueous solution, which was extracted with n-hexane-ether at a ratio of 2:1. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain 27.7 g of pure EPA. This compound was then structurally characterized using nuclear magnetic resonance ( 1 H NMR, 13 C NMR), and the results were consistent with those of Example 1 above.

[0039] 3. Esterification of EPA:

[0040] 27.7 g of the above-mentioned pure EPA was added to 85 mL of anhydrous ethanol, and 1.5 mL of concentrated sulfuric acid was slowly added dropwise to the solution under continuous stirring. Subsequently, the reaction was carried out at 70 °C for 4 hours, and after completion, it was naturally cooled to room temperature. Subsequently, the system was neutralized to pH = 7 using 2 M sodium hydroxide solution, and then the organic phase was washed with distilled water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product of EPA ethyl ester. Subsequently, molecular distillation was carried out to obtain 26.9 g of high-purity EPA-EE. This compound was then structurally characterized using nuclear magnetic resonance ( 1 HNMR, 13 C NMR), and the results were consistent with those shown in Example 1.

[0041] The present invention provides a large-scale EPA and DHA separation method based on the commonly used industrial preparation means without the need for chromatography. Compared with the prior art, the technical solution of the present invention can achieve efficient separation of EPA and DHA under conventional industrial equipment and prepare a high-purity EPA product; in addition, by further ethyl esterification of EPA, not only its stability and bioavailability are significantly improved, but also the separation cost is greatly reduced, and the production efficiency is improved, so that it is more widely used in medicines and health products. The present invention not only significantly reduces the separation cost, but also improves the production efficiency, and has broad market prospects and application value.

[0042] The technical solution of the present invention is described above in conjunction with specific implementation methods, but it should be noted that the above descriptions are only for explaining the solution of the present invention and cannot be interpreted in any way as a specific limitation on the scope of protection of the invention. Based on the explanation here, those skilled in the art can think of other specific implementation methods or equivalent replacements of the present invention without creative work, and they will all fall within the scope of protection of the present invention.

Claims

1. A method for large-scale separation and ethyl esterification of EPA in fish oil, characterized in that, The method includes: S1: Preparation of iodolactonide Dissolve the polyunsaturated fatty acid mixture in tetrahydrofuran and continuously stir. Subsequently, add sodium bicarbonate and an aqueous solution of sodium iodide to the above mixture and continue stirring. Then, continue to add elemental iodine, and iodolactone 3 and iodolactone 4 are formed by reaction. Subsequently, weigh a certain amount of sodium bicarbonate and a certain amount of sodium sulfite and dissolve them in distilled water to form a reaction solution. Slowly pour the above mixture into the reaction solution to quench the reaction. The obtained mixture is extracted with ethyl acetate. The organic phase is distilled under reduced pressure to obtain a mixture containing iodolactone 3 and iodolactone 4. The mixture is continuously dissolved in n-hexane, washed with a 0.5M sodium carbonate alkaline solution. The separated n-hexane is washed with saturated brine, dried over anhydrous sodium sulfate, and then distilled under reduced pressure. The obtained solution passes through a sintered funnel with a silica gel powder layer spread on the surface. After the filtrate is distilled under reduced pressure, refined iodolactone 3 and iodolactone 4 are obtained. S2: Purification of EPA Under nitrogen protection, add the above refined iodolactone 3 and iodolactone 4 to an acetonitrile solution containing sodium iodide or a chloroform solution containing sodium iodide. Subsequently, add triethylsilane and stir at room temperature. After the reaction ends, weigh a certain amount of sodium sulfite and a certain amount of sodium citrate and dissolve them in an aqueous solution. Add this solution to the solution after the above reaction ends to terminate the reaction. Subsequently, extract with n-hexane-dichloromethane. The extract is washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The obtained concentrated solution is dissolved in n-hexane. Subsequently, extract with a 0.5M sodium carbonate solution, wash with a 1:1 n-hexane-ethyl ether solution. After the washing ends, acidify with 4.5M hydrochloric acid, then extract with a 2:1 n-hexane-ethyl ether, and then wash with saturated brine and dry over anhydrous sodium sulfate, and then concentrate under reduced pressure to obtain pure EPA. The purified pure EPA is subjected to structural identification by NMR. S3: Esterification of EPA Add the above pure EPA to a certain amount of anhydrous ethanol. Slowly drop a small amount of concentrated sulfuric acid under continuous stirring, control the reaction temperature and time. After the reaction ends, cool to room temperature. Then, adjust the pH value to 7 with a 2M sodium hydroxide solution. Wash the organic phase with distilled water, dry over anhydrous sodium sulfate, and then concentrate under reduced pressure to obtain a crude product of EPA ethyl ester. Subsequently, perform molecular distillation to obtain high-purity EPA-EE, and perform structural identification on it by NMR.

2. The method for large-scale separation and ethyl esterification of EPA in fish oil according to claim 1, characterized in that, The preparation method of the aqueous sodium iodide solution includes: dissolving 110 - 170 g of sodium iodide in 850 - 1300 mL of distilled water and stirring and mixing.

3. A method for large-scale separation and ethyl esterification of EPA from fish oil according to claim 1, characterized in that, The sodium carbonate alkaline solution in step S1 and the sodium carbonate solution in step S2 both include 1:1 methanol and water.

4. A method for large-scale separation and ethyl esterification of EPA in fish oil according to claim 1, characterized in that, In step S3, control the reaction temperature to be 60°C - 70°C, react for 3 - 5 hours, and then cool to room temperature.

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